Lin W W, Wang C W, Chuang D M
Department of Pharmacology, National Taiwan University, Taipei.
J Neurochem. 1997 Jun;68(6):2577-86. doi: 10.1046/j.1471-4159.1997.68062577.x.
Primary cultures of cerebellar granule cells (CGCs) grown in high-K+ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3-4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC alpha, gamma, mu, lambda, and iota from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC epsilon and betaII, respectively, whereas no significant change was observed for betaI. The isoforms of delta, theta, eta, and zeta were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC iota and epsilon were decreased, those of PKC alpha, betaI, betaII, gamma, and lambda were unchanged, whereas that of PKC mu was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2-4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K+ cultures, levels of PKC alpha, mu, lambda, and iota, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC gamma, lambda, iota, and mu at 10 and 14 DIV. In addition, the PKC epsilon level was increased at 14 DIV but decreased at early stages, whereas PKC alpha, betaI, and betaII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up-regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK-801.
在高钾（25 mM；K25）培养基中培养的小脑颗粒细胞（CGCs）原代培养物在体外逐渐分化。体外培养3 - 4天（DIV）后分化明显，8 DIV后达到成熟阶段。CGCs培养时间延长（>13 DIV）会引发自发细胞死亡过程。然而，若在正常生理钾浓度（5 mM；K5）下培养，相当一部分细胞在培养第一周结束时死亡。为研究蛋白激酶C（PKC）在CGCs发育中的作用，我们测量了激酶活性以及激酶同工型的蛋白水平。随着K25 CGC培养的进行，PKC活性随时间依赖性增加3.2倍，在8 DIV时达到稳定状态。使用PKC同工型特异性抗体的蛋白质印迹分析显示，从2到8 DIV，PKCα、γ、μ、λ和ι的水平增加。PKCε和βII在4 DIV时分别略有增加或减少，而βI未观察到显著变化。未检测到δ、θ、η和ζ同工型。将14 DIV培养物与10 DIV培养物比较，PKCι和ε的免疫反应性降低，PKCα、βI、βII、γ和λ的免疫反应性未改变，而PKCμ的免疫反应性仍增加。在K5培养物中，2 - 4 DIV时每种PKC同工型的免疫反应性与K25细胞中观察到的相似，尽管未观察到明显的分化特征。与低钾培养物中8 DIV时细胞死亡的出现相协调，PKCα、μ、λ和ι的水平显著降低，而其他的未降低。NMDA受体拮抗剂MK - 801和2 - 氨基 - 5 - 磷酸戊酸显著阻止了CGCs的年龄诱导凋亡，在这些条件下细胞存活>18 DIV。MK - 801的细胞保护作用与10和14 DIV时PKCγ、λ、ι和μ水平的增加相伴。此外，与单独的K25培养相比，PKCε水平在14 DIV时增加但在早期降低，而PKCα、βI和βII水平未改变。综上所述，PKC同工型的诱导和上调可能在通过去极化和MK - 801维持CGCs存活中起重要作用。
